Constitutive expression of recombinant human hyaluronidase PH20 by Pichia pastoris

doi:10.1016/j.jbiosc.2016.06.007

Journal of Bioscience and Bioengineering

Volume 122, Issue 6, December 2016, Pages 673-678

https://doi.org/10.1016/j.jbiosc.2016.06.007 Get rights and content

PH20 is known as sperm adhesion molecule 1 (SPAM1) and also has hyaluronidase function to preferentially hydrolyze the glycosidic linkage of hyaluronic acid (HA). A DNA fragment containing core domain of human PH20 gene was cloned into a constitutive expression plasmid (pGAPZαC) of Pichia pastoris to produce a fusion protein with α factor signal in the N-terminus and 6 × His as well as c-Myc tags in the C-terminus. The resulting plasmid pGAPZαC-PH20 was integrated into the genome of P. pastoris strain GS115. Functional recombinant human PH20 (rHuPH20) was successfully expressed and secreted by the recombinant P. pastoris transformant. Highest hyaluronidase activity of 2 mU/mL could be obtained at 3 day in an YPD culture. After purified by phenylboronic acid resin adsorption, rHuPH20 with a specific activity of 230 mU/mg was obtained. Via periodic acid-Schiff staining and zymogram analysis, the partially purified rHuPH20 was determined to be highly glycosylated to various extents with molecular mass in the range of 100–300 kDa. The enzyme showed a maximal activity at pH 5.0 but no appreciable activity at pH ≤3 and pH ≥8. The hyaluronidase activity could be stably maintained at 4°C but lost 40% after incubating at 30°C for 4 h. Both N-acetyl cysteine and glutathione showed a half maximal inhibitory concentration (IC₅₀) of 8 mM against rHuPH20.

Section snippets

Materials

All materials and chemicals used were analytical grade. The plasmid pMW172 harboring human PH20 gene was kindly provided by Professor Günter Lepperdinger at Institute for Biomedical Aging Research, Austrian Academy of Sciences (Innsbruck, Austria). Sharp Protein Markers III was obtained from Yeastern Biotech Co., Ltd. (Taipei, Taiwan). Sodium hyaluronate (HA) was obtained from First Cosmetics Manufacture Co. (Taipei, Taiwan). The expression host P. pastoris strain GS115 and vector pGAPZαC were

Growth curves of rHuPH20 expressing cells

In addition to the commonly used Pichia growth medium BMGY, the yeast growth medium YPD was also employed to culture the selected transformant P. pastoris GS115/PH20 for the production of rHuPH20. As shown in Fig. 1, initially the cells grew very fast in both BMGY and YPD medium. Cell density could reach OD 35 after 1 day. However, the growth rate started to decrease significantly in the following days, probably due to the additional energy burden to drive the constitutive expression of

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Constitutive expression of recombinant human hyaluronidase PH20 by Pichia pastoris

Section snippets

Materials

Growth curves of rHuPH20 expressing cells

J. Biol. Chem.

Microvasc. Res.

Matrix Biol.

Eur. J. Pharm. Sci.

FEBS Lett.

FEBS Lett.

J. Control. Release

Fertil. Steril.

FEMS Microbiol. Rev.

Int. J. Biol. Macromol.

Carbohydr. Polym.

J. Biol. Chem.

Biochem. Eng. J.

Gene

Biochem. Biophys. Res. Commun.

Enzyme Microb. Technol.

Anal. Biochem.

Inhibitory effects of hydrangenol derivatives on the activation of hyaluronidase and their antiallergic activities

Planta Med.

Indomethacin as inhibitor of hyaluronidase

Arch. Immunol. Ther. Exp.

Hyaluronate in vasculogenesis

Science